The overall aim of this Program Project is to conduct integrated, interdisciplinary studies of the mechanisms of pro- and anti-apoptotic signaling in hepatocytes, especially the signaling pathways activated by TNFalpha and Fas that lead to mitochondrial permeabilization and release of cytochrome c. In Project 1, molecular biology approaches will be used to elucidate how proteins whose synthesis is under the control of NFkappaB act to regulate upstream apoptotic signals that converge to induce the mitochondrial permeability transition (MPT). Project 2 will address the cell biological aspects of the MPT, specifically how the MPT triggers mitochondria autophagy and how dysregulated can lead to apoptosis. Finally, Project 4 examines the cellular physiology of apoptosis with special reference to the mitochondrial changes that precede and promote onset of the MPT after pro-apoptotic signaling. These 4 projects are supported 5 cores: a Gene Delivery Core to prepare adenoviral gene vectors, a Cell and Molecular Imaging Core for confocal and multiphoton microscopy, a Lipid/Ceramide Core to perform chemical analysis and synthesis of sphingolipid metabolites and analogs, a Cell Isolation/Transgenic Core to isolate hepatocytes and maintain transgenic strains of mice, and an Administrative Core. Together the projects and cores are designed to provide a comprehensive picture of apoptotic signaling from cell surface ligand-receptor interaction to mitochondrial permeabilization. Our overall approach integrate cell, molecular, biophysical and biochemical approaches, and the Project will generate fundamental new information relevant to TNFalpha-and Fas-induced apoptotic-signaling in liver disease.